Branched chain ketoacid dehydrogenase is an unusual dehydrogenase which catalyzes the oxidative decarboxylation of branched chain ketoacid to their corresponding acyl-coenzyme A esters. This enzyme is defective in the genetic disease, maple syrup urine disease resulting in accumulation of toxic levels of the branched chain ketoacids causing mental retardation and death. The probability is high that the native enzyme is a complex, similar to pyruvate dehydrogenase, consisting of decarboxylase, transacetylase and dihydrolipoyl dehydrogenase. We believe that the branched chain ketoacid dehydrogenase of Pseudomonas putida will be a good model for the study of branched chain ketoacid dehydrogenase of man. We have purified branched chain ketoacid dehydrogenase from P. putida 30 fold using an assay which measures the activity of the entire complex. Branched chain amino acids are activators of the enzyme, an interesting regulatory property which appears to be unique for amino acid catabolic pathways. Because branched chain ketoacid dehydrogenase is the first enzyme common to the metabolism of both D and L-valine, it is in a sensitive position to control the flow of metabolites through the pathway, and its activation by branched chain amino acids may have important physiological consequences. We plan to purify the intact complex and to study its catalysis, regulation and organization. We plan to separate the subunits, determine their molecular weight, amino acid composition and catalytic properties. We will study the organization of the complex by electron microscopy in order to determine the unit cell dimensions. We have several mutants with defective branched chain ketoacid dehydrogenase and the location of the mutations will be mapped using the P. putida K factor. The fine structure of that region of the chromosome which contains the genes for branched chain ketoacid dehydrogenase will be studied by transduction and these results will be used to confirm the structure for the enzyme developed from the preceding studies.